The regulation of the cell cycle in plants is poorly understood. Most of the knowledge regarding the regulation of DNA replication, also known as the S-phase of the cell cycle regulation originates from experimental data obtained in yeast and mammalian cells. However, the importance to understand the cell cycle regulation in plant cells has become increasingly important in agriculture, e.g. to control growth of plants at stress conditions, to obtain resistance against parasites that block or modulate the cell cycle regulation, or to improve the yield of agriculturally important crops. Further, one might be interested to intervene in the cell cycle regulation by allowing further rounds of DNA replication, but simultaneously preventing further cell cycle progress by blocking the subsequent mitosis. In this way, cells may be obtained having multiple sets of their genetic material, so that plants with a high rate of endoreduplication may be generated. The term “endoreduplication” means recurrent DNA replication without consequent mitosis and cytokinesis.
From experiments in yeast, it is known that DNA replication and mitosis are coupled events in the cell cycle. Paulovich et al., 1997; Cell 88, 315-321. Genetic studies in yeast for example suggest that the CDC7 serine-threonine kinase plays a role in the initiation of DNA synthesis. Evidence has been presented that CDC7 is apparently directly involved in the activation of individual early—as well as late replication origins during S-phase (Bousset and Diffley, 1998, Genes Dev 12, 480-490; Donaldson et al., 1998, Genes Dev 12, 491-501). The protein levels of CDC7 are constant during the cell cycle.
Activation of CDC7 as a kinase occurs at the G1/S transition of the cell cycle and is dependent on the binding with another factor, DBF4, at the G1/S transition of the cell cycle, probably by phosphorylating proteins at the origins (Kitada et al, 1992; Genetics 131: 21-29, Lei et al; Genes and Development 11, 3365-3374, 1997). In order to function as a kinase, the CDC7 kinase may be a substrate for one or more phosphorylation events. Overexpressed kinase-negative mutants of CDC7 arrest yeast cells in the G1 to S transition and inhibit growth. Further experiments showed that the inactivation of wild-type CDC7 function probably can be explained through titration of DBF4 by the inactive cdc7 mutant proteins (Ohtoshi et al., 1997, Mol Gen Genet 254, 562-570). In addition to mechanisms to control the onset of DNA replication, other mechanisms contribute to restrict DNA replication to occur only once during the cell cycle. For example, the CDC16, CDC23 and CDC27 proteins are part of a high molecular weight complex, known as the anaphase promoting complex (APC) or cyclosome, (see Romanowski and Madine, Trends in Cell Biology 6, 184-188, 1996, and Wuarin and Nurse, Cell 85, 785-787 (1996), both incorporated herein by reference). The complex in yeast is composed of at least 8 proteins, the TPR (tetratricopeptide repeat) containing proteins CDC16, CDC23 and CDC27, and five other subunits named APC1, APC2, APC4, APC5 and APC7 (Peters et al. 1996, Science 274, 1199-1201). The APC targets its substrates for proteolytic degradation by catalyzing the ligation of ubiquitin molecules to these substrates. APC-dependent proteolysis is required for the separation of the sister chromatids at meta—to anaphase transition and for the final exit from mitosis. Among the APC-substrates are the anaphase inhibitor protein Pds1p and mitotic cyclins such as cyclin B, respectively (Ciosk et al. 1998, Cell 93, 1067-1076; Cohen-Fix et al. 1996, Genes Dev 10, 3081-3093; Sudakin et al. 1995, Mol Biol Cell 6, 185-198; Jorgensen et al. 1998, Mol Cell Biol 18, 468-476; Townsley and Ruderman 1998, Trends Cell Biol 8, 238-244). To become active as a ubiquitin-ligase, at least CDC16, CDC23 and CDC27 need to be phosphorylated in the M-phase (Ollendorf and Donoghue 1997, J Biol Chem 272, 32011-32018). Activated APC persists throughout Gl of the subsequent cell cycle to prevent premature appearance of B-type cyclins which would result in an uncontrolled entry into S-phase (Irniger and Nasmyth 1997, J Cell Sci 110, 1523-1531). It has been demonstrated in yeast that mutations in either of at least two of the APC components, CDC16 and CDC27, can result in DNA overreplication without intervening passages through M-phases (Heichman and Roberts 1996, Cell 85, 39-48). CDC16, CDC23 and CDC27 all are tetratricopeptide repeat (TPR) containing proteins. A suggested minimal consensus sequence of the TPR motif is as follows: X3—W—X2-L-G-X2—Y—X8-A-X3—F—X2-A-X4—P—X2 (Lamb et al. 1994, EMBO J 13, 4321-4328; X denotes any amino acid, Xn a stretch of n of such amino acids). However, the consensus residues can exhibit significant degeneracy and little or no homology is present in non-consensus residues. The hydrophobicity and size of the consensus residues, rather than their identity, seems to be important. TPR motifs are present in a wide variety of proteins functional in yeast and higher eukaryotes in mitosis (including the APC protein components CDC16, CDC23 and CDC27), transcription, splicing, protein import and neurogenesis (Goebl and Yanagida 1991, Trends Biochem Sci 16, 173-177). The TPR forms a α helical structure, tandem repeats organize into a superhelical structure ideally suited as interfaces for protein recognition (Groves and Barford 1999, Curr Opin Struct Biol 9, 383-389). Within the α helix, two amphipathic domains are usually present, one at the NH2-terminus and the other near the COOH-terminus (Sikorski et al. 1990, Cell 60, 307-317).